The goal of this proposal is to develop a new strategy that combines experiment and theory for rapidly characterizing the binding modes and activity of small molecules that target flexible RNA receptors and to apply this strategy in the discovery of lead compounds that bind and disrupt the activity of two major HIV RNA drug targets. HIV/AIDS is currently the world's most urgent public health challenge. While antiretroviral therapies have significantly improved the prognosis for HIV infection, there is a growing need for antiviral agents that target new viral components and thereby further suppress the rate of replication and resistance. Genomes of all RNA viruses, such as HIV, contain essential RNA structures that can widen drug-targets making it possible to inhibit new steps in the viral life cycle for which we do not currently have druggable protein targets. Efforts to find small molecules that target regulatory RNAs have been hindered by lack of suitable methods for efficiently screening RNAs that lack the necessary readout enzymatic activity. Computational docking methods can, in principle, overcome many of the limitations inherent to experimental methods and can provide the structural and dynamical information needed to assess small molecule activity. However, current docking protocols fail to take into account the very large changes in structure that flexible RNA receptors typically undergo on binding small molecules. There is growing evidence that small molecules bind pre-existing RNA conformers from a dynamical ensemble; thus, if the structures of the unbound RNA dynamical ensembles could be determined at atomic resolution, a major obstacle to computational docking could be overcome. We propose to develop a new method, that combines NMR spectroscopy and enhanced computational MD simulations, to visualize at atomic resolution, unbound RNA structural ensembles in free and protein/metal bound states and to use virtual docking simulations to identity small molecules that bind distinct conformers in the ensemble. The new methodology we will be used to identify small molecules that disrupt (i) viral transcription elongation by targeting the transactivation response element (TAR) RNA and (ii) viral genome dimerization, packaging, and maturation by targeting the dimerization initation site (DIS) RNA. Computational predictions will be experimentally verified and complemented by in vitro NMR, florescence, and reporter gene assays as well as in vivo viral replication assays. This unique blend of experiment and theory will help establish a new paradigm for RNA- targeted HIV drug discovery.